A primer and crRNA for detection of oropouche virus nucleic acid, a kit and a detection method

Abstract

The application aims to provide a primer and crRNA, a kit and a detection method for detection of oropouche virus nucleic acid, which are suitable for detection in places with insufficient resources, have short detection time, good specificity, high sensitivity, do not need to depend on a fluorescent PCR instrument, and can observe the detection result by naked eye through irradiation of 470nm blue light. The application comprises a primer and a crRNA, wherein the primer comprises a forward primer and a reverse primer. The application is applied to the technical field of virus nucleic acid detection.

Description

Technical Field

[0001] This invention relates to the technical field of viral nucleic acid detection, and particularly to primers and crRNA, a reagent kit, and a detection method for oropeceutical virus nucleic acid detection. Background Technology

[0002] Oropouche virus (OROV) belongs to the order Bunyavirales, family Peribunyaviridae. It is an arbovirus transmitted by Culicoides paraensis and mosquitoes, causing Oropouche fever (OROF). Clinical manifestations include fever, headache, myalgia, and joint pain, similar to the symptoms of dengue fever and Zika virus infection, often leading to misdiagnosis or missed diagnosis. Since its initial discovery in Trinidad and Tobago in 1955, OROV has caused numerous outbreaks in several Central and South American countries, including Brazil, Peru, Colombia, and Bolivia, with over 500,000 reported cases. In recent years, the prevalence of OROV has been expanding, with local or imported cases reported in Brazil, Cuba, Italy, and other countries in 2024, indicating an increasing risk of cross-border transmission. Although no imported cases have been reported in my country, the risk of Orovirus transmission cannot be ignored due to frequent international exchanges and climate change. It is urgent to establish rapid and sensitive testing methods to strengthen port monitoring capabilities.

[0003] Currently, the mainstream detection technologies for oropic prophylaxis virus mainly include serological testing and polymerase chain reaction (PCR) testing. Serological testing has significant limitations in screening cases in the early stages of viral infection, typically requiring 5-7 days after the onset of symptoms to detect specific antibodies. Furthermore, this method is prone to cross-reactivity, affecting the accuracy of test results. Nucleic acid testing has gained considerable attention due to its high sensitivity and ability to detect the virus in the early stages of the disease. Currently, PCR testing is the primary method for its application. However, this technology has specific requirements for experimental equipment, requiring specialized electrophoresis apparatus, quantitative PCR instruments, etc. It also demands high standards for the cleanliness of the operating environment and the professional skills of the operators. In addition, the testing process is time-consuming. These factors collectively limit the practical application of PCR testing.

[0004] For example, Chinese patent CN119119302A discloses an oropeceutical virus fusion protein, its preparation method, and its application. By detecting these specific antibodies in serum, it is possible to determine whether a subject is infected with the virus. However, this detection method is time-consuming, has low sensitivity, and requires specific experimental equipment, such as professional electrophoresis apparatus and quantitative real-time PCR instruments. It also places high demands on the cleanliness of the operating environment and the professional skills of the operators. Therefore, it is necessary to provide primers and crRNA, a reagent kit, and a detection method for oropeceutical virus nucleic acid detection that is time-efficient, highly specific, and sensitive. This method does not require a fluorescence PCR instrument; the results can be observed visually with only 470nm blue light irradiation, making it suitable for detection in resource-constrained areas. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the shortcomings of the prior art and provide primers and crRNA, kits and detection methods for the detection of oropeceutical virus nucleic acid. The detection is time-saving, specific and sensitive. It does not require a fluorescence PCR instrument and only requires 470nm blue light irradiation to observe the detection results with the naked eye. It is suitable for detection in places with limited resources.

[0006] The technical solution adopted in this invention is as follows: This invention includes primers and crRNA, wherein the primers include a forward primer and a reverse primer, and the base sequences of the forward primer, the reverse primer, and the crRNA are respectively represented as follows: The forward primer: OROV-F:5'-TAATACGACTCACTATAGGGTGCTGAGATGTTTCTGCAGACATTCACTTTC-3', SEQ ID NO: 1, its position in GenBank reference sequence KP691605.1 is 479~509; The reverse primer: OROV-R: 5'-TGCTCTTACAGCAACTATTTCTTCACGCATCC-3', SEQ ID NO: 2, its position in GenBank reference sequence KP691605.1 is 616~647; The crRNA: 5-gauuuagacuaccccaaaaacgaaggggacuaaaacGCAGUCUUCUGGCCAUACCUCUGCCGAA-3', SEQ ID NO: 3, is located at position 580~607 in GenBank reference sequence KP691605.1.

[0007] As can be seen from the above scheme, this application provides primers and crRNA sequences for the detection of oropeceutical virus nucleic acid. Using these primers and crRNA, a kit for the detection of oropeceutical virus with high repeatability, high specificity, and high sensitivity is achieved. The detection time is short, requiring only 40 minutes. It exhibits good specificity, high sensitivity, strong anti-interference ability, simple operation, and wider applicability, making it suitable for early detection and case finding of oropeceutical virus infection, thus better controlling the virus. As a simplified result interpretation method, this application does not rely on a fluorescence PCR instrument; only 470nm blue light irradiation is needed for visual observation of the test results, making it suitable for testing in resource-limited areas. This application can be applied to routine testing and disease control in clinical settings and ports of entry.

[0008] In a preferred embodiment, the kit comprises RPA reaction reagents and CRISPR reaction reagents; The RPA reaction reagent includes buffer A, 20 μM primer OROV-F, 20 μM primer OROV-R, starter, and reaction tube, wherein primer OROV-F is the nucleotide sequence shown in SEQ ID NO: 1, and primer OROV-R is the nucleotide sequence shown in SEQ ID NO: 2; The CRISPR reaction reagent includes Cleavage Buffer, Trans Mix, T7RNA Polymerase, Cas13a Protein (2uM), crRNA (0.4uM), Reporter (4uM), and Nuclease-free Water, wherein the crRNA is the nucleotide sequence shown in SEQ ID NO:3.

[0009] A preferred embodiment is that the kit comprises RPA reaction reagent and CRISPR reaction reagent, characterized in that: the positive control is an RNA fragment transcribed in vitro from oropic virus.

[0010] A preferred approach is to use sterile physiological saline as the negative control, which is extracted simultaneously with the sample during nucleic acid extraction; and to use ultrapure water without nuclease as the blank control.

[0011] A preferred embodiment is that the method for preparing the positive control includes the following steps: Step A1: Synthesize plasmid based on reference sequence KP691605.1 downloaded from GenBank; Step A2: Use T7 RNA polymerase to perform in vitro transcription to obtain single-stranded RNA, and then digest with DNase I to remove the DNA molecules. Step A3: Perform column purification using the QIAGEN RNeasy MiniElute Cleanup kit; Step A4: Determine the concentration of purified RNA using a micro-ultraviolet spectrophotometer, and calculate the copy number based on its molecular weight; Step A5: After aliquoting, store at -80℃ as a positive control for the kit.

[0012] A preferred embodiment is that the detection method includes the following steps: Step B1: Extract RNA from the sample, i.e. template RNA. The kit does not provide RNA sample extraction reagents. Users should select the appropriate commercial kit to extract viral nucleic acid according to the sample type. Step B2: Prepare the RPA reaction solution as follows: The reaction volume is 50 μL; add the reaction mixture to each reaction tube containing 29.4 μL of buffer A, 0.4 μM of each primer (OROV-F, OROV-R), 2.5 μL of starter, and 10 μL of RNA template; mix well, centrifuge for a few seconds, and place in a water bath, metal bath, or PCR instrument at 39℃ for 10 min; Step B3: Prepare CRISPR reaction solution for instrument detection: The reaction volume is 20 μL; the mixture contains 2 μL Cleavage Buffer, 4 μL Trans Mix, 0.5 μL T7 RNA Polymerase, 0.6 μL Cas13a Protein (2 μM), 1.5 μL crRNA (0.4 μM), 0.6 μL Reporter (4 μM), 5 μL of the product from the first reaction, and 5.8 μL Nuclease-free Water. After mixing, place the mixture on a fluorescence PCR instrument at 37℃ for 25 min, and collect the fluorescence of the FAM channel once per minute. Step B4, Result Judgment: First, the quality control system should be judged, that is, the amplification curve of the positive control in each fluorescence channel should show a standard S-shaped curve, while the negative control and blank control should not show a typical S-shaped curve. When the quality control system is effective, the results of the test samples are judged as follows: if the test sample has a clear S-shaped curve in the FAM channel, it is judged as positive for oropeceutical virus nucleic acid; if the test sample has no fluorescence amplification in the FAM channel, it is judged as negative for oropeceutical virus nucleic acid.

[0013] In a preferred embodiment, the detection method includes sensitivity analysis. The method for sensitivity analysis is as follows: (1) Sample processing: Using the viral RNA transcribed in vitro as the template for detection, the concentration of purified RNA was determined using a micro-ultraviolet spectrophotometer. The copy number of the initial RNA template was calculated based on the molecular weight. Then, it was serially diluted 10-fold to single-digit copy numbers, for a total of 5 gradients. The copy number was 9 × 10⁻⁶. 2 copies / μL~10 -1 copies / μL; (2) Detection: The reaction solution was prepared and detected using the detection method described above, and the minimum template copy number that the kit can detect was analyzed. (3) Results: The test results show that the detection limit of the kit is 9 copies / μL and the total detection time is 40 min. Therefore, the kit has a fast detection speed and high sensitivity, and can play an important role in rapid emergency detection.

[0014] In a preferred embodiment, the detection method includes repeatability analysis. The method for repeatability analysis is as follows: (1) Sample processing: The viral RNA transcribed in vitro was used as the template for detection at a concentration of 9 copies / μL; (2) Detection: The reaction solution was prepared using the detection method described above, and the detection was repeated 10 times. Then, the reaction tube was placed on a fluorescence PCR instrument for detection. After the reaction was completed, the fluorescence curve was observed, and the repeatability of the detection by the kit was analyzed. (3) Results: The test results showed that the kit was positive in 10 replicates at a concentration of 9 copies / μL, which was in line with the expected results. It can be seen that the test kit has good repeatability.

[0015] In a preferred embodiment, the detection method includes specificity analysis. The method for the specificity analysis is as follows: (1) Sample processing: Select a group of clinically positive samples of bloodborne infectious disease-related pathogens, including dengue virus type I, dengue virus type II, dengue virus type III, dengue virus type IV, chikungunya virus, Zika virus, yellow fever virus, monkeypox virus, Plasmodium falciparum, and Plasmodium vivax, plus in vitro transcribed oropecie virus RNA (10 4 (copies / μL) (2) Detection: Using the test kit and the method described above, the 11 samples were tested to observe whether the test kit would produce non-specific test results; (3) Results: Based on the analysis of the fluorescence spectrum amplified by the detection kit, the kit was positive only for samples containing oropic virus RNA, while the detection of the other 10 pathogens and the negative control was negative, proving that the method has good specificity.

[0016] A preferred approach is to analyze the sensitivity and repeatability of a kit for detecting oropic virus based on RPA-CRISPR / Cas13a technology using a visualized result interpretation method: (1) Sample processing: Copy number: 9 × 10 2 copies / μL~10 -1 OROVRNA at a concentration of copies / μL was used as a template; (2) Detection: Using the kit, place the CRISPR reaction tube in a 37°C constant temperature water bath for 25 min. Finally, observe the color and interpret the result under 470nm blue light. Yellow-green fluorescent tubes are positive, and those without fluorescence are negative. Repeat the test 20 times with the lowest detection concentration of the template to determine its repeatability. (3) Results: According to the detection results, the lowest concentration of the sample emitting yellow-green fluorescence was 9 copies / μL, and all 20 repeated tests were positive; indicating that the detection limit of the kit under the visualization result interpretation method is 9 copies / μL. It can be seen that the detection kit is fast, highly sensitive, and does not depend on the fluorescence PCR instrument, and can play an important role in rapid emergency detection in areas with scarce resources. Attached Figure Description

[0017] Figure 1 This invention is applied to sensitivity analysis, with values ​​of 9×10 from top to bottom. 2 copies / μL~10 -1 Detection results of copies / μL oropeceutical virus RNA samples; Figure 2 This invention is applied to repeatability analysis, with 10 curves representing the detection results of 10 replicates of a 9 copies / μL oropekee virus RNA sample; Figure 3 When this invention was applied to specific analysis, the test was repeated three times. Only the OROV sample showed an amplification curve, while the other 10 arboviruses did not show an amplification curve. Figure 4 This invention is applied to anti-interference analysis, and the results were still positive in three repeated tests in samples of hemolysis, lipemia, ibuprofen, and acetaminophen. Figure 5 This invention is applied to sensitivity analysis in a visual result interpretation method. Positive samples show visible yellow-green fluorescence under blue light at a wavelength of 470nm, while negative samples show no fluorescence. Figure 6 This invention is applied to repeatability analysis under the visualization result interpretation method. Positive samples (1~20) with 9 copies / μL showed visible yellow-green fluorescence under blue light irradiation at a wavelength of 470nm, while negative samples (21) showed no fluorescence. Detailed Implementation

[0018] Example 1 Specific primer design: By collecting 320 oropecene virus S genome sequences from GenBank and performing homology alignment, key elements such as variable regions, conserved regions, and specific regions were analyzed. Generally, RPA primers are 30-35 bp in length, and ideally, no special sequences such as long polypurine or polypyrimidine chains should appear in the primers. The GC content is preferably between 40% and 60%, and the crRNA design should not overlap with the RPA primers. Multiple pairs of RPA primers and crRNAs were designed based on these principles. After synthesizing the designed primers, they were screened and verified using samples. Finally, the following RPA primers and crRNAs were selected as the preferred primers in this invention.

[0019] This invention includes primers and crRNA, characterized in that the primers include a forward primer and a reverse primer, and the base sequences of the forward primer, the reverse primer, and the crRNA are respectively represented as follows: The forward primer: OROV-F:5'-TAATACGACTCACTATAGGGTGCTGAGATGTTTCTGCAGACATTCACTTTC-3', SEQ ID NO: 1, its position in GenBank reference sequence KP691605.1 is 479~509; The reverse primer: OROV-R: 5'-TGCTCTTACAGCAACTATTTCTTCACGCATCC-3', SEQ ID NO: 2, its position in GenBank reference sequence KP691605.1 is 616~647; The crRNA: 5-gauuuagacuaccccaaaaacgaaggggacuaaaacGCAGUCUUCUGGCCAUACCUCUGCCGAA-3', SEQ ID NO: 3, is located at position 580~607 in GenBank reference sequence KP691605.1.

[0020] In this embodiment, the genome sequences of oropic viruses collected from GenBank were collected and homology comparisons were performed to analyze key elements such as variable regions, conserved regions, and specific regions. Generally, RPA primers are 30-35 bp in length, and ideally, no special sequences such as long polypurine or polypyrimidine chains should appear in the primers. The GC content is preferably between 40% and 60%, and the design of crRNA should not overlap with the RPA primers. Multiple pairs of RPA primers and crRNAs were designed based on these principles, and suitable RPA primers and crRNAs were screened. The concentrations of primers, crRNA, and enzymes were adjusted and optimized to establish an RPA-CRISPR / Cas13a reaction system. The first reaction mixture was added to each reaction tube tube with 29.4 μL of buffer A, 0.4 μM of each primer (OROV-F, OROV-R), 2.5 μL of starter, and 10 μL of RNA template. After mixing, the mixture was placed in a water bath, metal bath, or PCR instrument at 39°C for 15 min. The second reaction mixture contained 2 μL of Cleavage Buffer, 4 μL of Trans Mix, 0.5 μL of T7 RNA Polymerase, 0.6 μL of Cas13a Protein (2 μM), 1.5 μL of crRNA (0.4 μM), 0.6 μL of Reporter (4 μM), 5 μL of the product from the first reaction, and 5.8 μL of Nuclease-free Water. After mixing, the mixture was placed on a PCR instrument and incubated at 37°C for 25 min, with the fluorescence of the FAM channel collected every minute.

[0021] Example 2 Kit composition and detection method for oropic virus detection based on RPA-CRISPR / Cas13a technology: The kit includes RPA reaction reagents and CRISPR reaction reagents; The RPA reaction reagent includes buffer A, 20 μM primer OROV-F, 20 μM primer OROV-R, starter, and reaction tube, wherein primer OROV-F is the nucleotide sequence shown in SEQ ID NO: 1, and primer OROV-R is the nucleotide sequence shown in SEQ ID NO: 2; The CRISPR reaction reagent includes Cleavage Buffer, Trans Mix, T7RNA Polymerase, Cas13a Protein (2uM), crRNA (0.4uM), Reporter (4uM), and Nuclease-free Water, wherein the crRNA is the nucleotide sequence shown in SEQ ID NO:3.

[0022] In this embodiment, the kit includes RPA reaction reagent and CRISPR reaction reagent, and the positive control is an RNA fragment transcribed from oropecie virus in vitro.

[0023] In this embodiment, the negative control is sterile physiological saline, which is extracted simultaneously with the sample during nucleic acid extraction to serve as a negative control; the blank control is ultrapure water without nuclease.

[0024] In this embodiment, the preparation method of the positive control includes the following steps: Step A1: Synthesize plasmid based on reference sequence KP691605.1 downloaded from GenBank; Step A2: Use T7 RNA polymerase to perform in vitro transcription to obtain single-stranded RNA, and then digest with DNase I to remove the DNA molecules. Step A3: Perform column purification using the QIAGEN RNeasy MiniElute Cleanup kit; Step A4: Determine the concentration of purified RNA using a micro-ultraviolet spectrophotometer, and calculate the copy number based on its molecular weight; Step A5: After aliquoting, store at -80℃ as a positive control for the kit.

[0025] In this embodiment, the detection method includes the following steps: Step B1: Extract RNA from the sample, i.e. template RNA. The kit does not provide RNA sample extraction reagents. Users should select the appropriate commercial kit to extract viral nucleic acid according to the sample type. Step B2: Prepare the RPA reaction solution as follows: The reaction volume is 50 μL; add the reaction mixture to each reaction tube containing 29.4 μL of buffer A, 0.4 μM of each primer (OROV-F, OROV-R), 2.5 μL of starter, and 10 μL of RNA template; mix well, centrifuge for a few seconds, and place in a water bath, metal bath, or PCR instrument at 39℃ for 10 min; Step B3: Prepare CRISPR reaction solution for instrument detection: The reaction volume is 20 μL; the mixture contains 2 μL Cleavage Buffer, 4 μL Trans Mix, 0.5 μL T7 RNA Polymerase, 0.6 μL Cas13a Protein (2 μM), 1.5 μL crRNA (0.4 μM), 0.6 μL Reporter (4 μM), 5 μL of the product from the first reaction, and 5.8 μL Nuclease-free Water. After mixing, place the mixture on a fluorescence PCR instrument at 37℃ for 25 min, and collect the fluorescence of the FAM channel once per minute. Step B4, Result Judgment: First, the quality control system should be judged, that is, the amplification curve of the positive control in each fluorescence channel should show a standard S-shaped curve, while the negative control and blank control should not show a typical S-shaped curve. When the quality control system is effective, the results of the test samples are judged as follows: if the test sample has a clear S-shaped curve in the FAM channel, it is judged as positive for oropeceutical virus nucleic acid; if the test sample has no fluorescence amplification in the FAM channel, it is judged as negative for oropeceutical virus nucleic acid.

[0026] Precautions: Powder-free gloves should be used throughout the entire experiment. To avoid cross-contamination during the experiment, when adding the template, add the blank and negative control first, then add the test sample, and finally add the positive control.

[0027] Example 3 Sensitivity analysis of kits for detecting oropic virus based on RPA-CRISPR / Cas13a technology, such as... Figure 1 As shown, the method for sensitivity analysis is as follows: 1) Sample processing: Using the viral RNA transcribed in vitro as a template for detection, the concentration of purified RNA was measured using a micro-ultraviolet spectrophotometer. The copy number of the initial RNA template was calculated based on its molecular weight. Then, it was serially diluted 10-fold to single-digit copy numbers, for a total of 5 dilutions, resulting in a copy number of 9 × 10⁻⁶. 2 copies / μL~10 -1 copies / μL; (2) Detection: The reaction solution was prepared and detected using the detection method described above, and the minimum template copy number that the kit can detect was analyzed. (3) Results: The test results show that the detection limit of the kit is 9 copies / μL and the total detection time is 40 min. Therefore, the kit has a fast detection speed and high sensitivity, and can play an important role in rapid emergency detection.

[0028] Example 4 Repeatability analysis of a kit for detecting oropic virus based on RPA-CRISPR / Cas13a technology, such as Figure 2 As shown, the method for repeatability analysis is as follows: The method for repeatability analysis is as follows: (1) Sample processing: The viral RNA transcribed in vitro was used as the template for detection at a concentration of 9 copies / μL; (2) Detection: The reaction solution was prepared using the detection method described above, and the detection was repeated 10 times. Then, the reaction tube was placed on a fluorescence PCR instrument for detection. After the reaction was completed, the fluorescence curve was observed, and the repeatability of the detection by the kit was analyzed. (3) Results: The test results showed that the kit was positive in 10 replicates at a concentration of 9 copies / μL, which met the expected results. It can be seen that the test kit has good repeatability.

[0029] Example 5 Specificity analysis of kits for detecting oropic virus based on RPA-CRISPR / Cas13a technology, such as... Figure 3 As shown, the method for the specificity analysis is as follows: (1) Sample processing: Select a group of clinically positive samples of bloodborne infectious disease-related pathogens, including dengue virus type I, dengue virus type II, dengue virus type III, dengue virus type IV, chikungunya virus, Zika virus, yellow fever virus, monkeypox virus, Plasmodium falciparum, and Plasmodium vivax, plus in vitro transcribed oropecie virus RNA (10 4 (copies / μL) (2) Detection: Using the test kit and the method described above, the 11 samples were tested to observe whether the test kit would produce non-specific test results; (2) Results: Based on the analysis of the fluorescence spectrum amplified by the detection kit, the kit was positive only for samples containing oropic virus RNA, while the detection of the other 10 pathogens and the negative control was negative, proving that the method has good specificity.

[0030] Example 6 like Figures 5 to 6 As shown, the sensitivity and repeatability analysis of the kit for detecting oropic virus based on RPA-CRISPR / Cas13a technology under the visualization result interpretation method are as follows: (1) Sample processing: Copy number: 9 × 10 2 copies / μL~10 -1 OROVRNA at a concentration of copies / μL was used as a template; (2) Detection: Using the kit, place the CRISPR reaction tube in a 37°C constant temperature water bath for 25 min. Finally, observe the color and interpret the result under 470nm blue light. Yellow-green fluorescent tubes are positive, and those without fluorescence are negative. Repeat the test 20 times with the lowest detection concentration of the template to determine its repeatability. (3) Results: According to the detection results, the lowest concentration of the sample emitting yellow-green fluorescence was 9 copies / μL, and all 20 repeated tests were positive; indicating that the detection limit of the kit under the visualization result interpretation method is 9 copies / μL. It can be seen that the detection kit is fast, highly sensitive, and does not depend on the fluorescence PCR instrument, and can play an important role in rapid emergency detection in areas with scarce resources.

[0031] like Figures 1 to 6 As shown, this application provides an evaluation of the sensitivity, repeatability, specificity, and anti-interference ability of the above-mentioned detection method. A primer, crRNA, and kit for oropeceutical virus nucleic acid detection provided in this application have the following advantages: short detection time (only 40 minutes); high specificity, high sensitivity, and strong anti-interference ability, making it suitable for early detection and case finding of oropeceutical virus infection, thus better controlling oropeceutical virus.

[0032] This application constructs an oropecten virus nucleic acid detection system based on RPA-CRISPR / Cas13a technology. CRISPR (clustered regularly interspaced short palindromic repeats) is a class of DNA sequence elements found in prokaryotes such as bacteria and archaea. The core components of the CRISPR-Cas system include a leader sequence, a CRISPR locus, and an associated gene encoding Cas (CRISPR-associated proteins). Its mechanism of action is as follows: after crRNA (CRISPRRNA) specifically targets and recognizes a foreign gene, it can activate the endonuclease activity of Cas proteins, thereby cleaving invading foreign genetic material and enabling prokaryotes to acquire immune defense functions. Since its discovery in 2011, researchers have successively identified a variety of new protein members. Currently, it is clear that the CRISPR / Cas system includes two major classes with more than 30 subtypes. Among them, proteins such as Cas9, Cas12, and Cas13 in the second class have shown broad application potential in the field of molecular detection due to their unique functional characteristics. To achieve the above objectives and other related application needs, this application provides specific primers and crRNA suitable for the detection of oropeceutical virus nucleic acid, and constructs a detection kit containing the above primers and crRNA. This kit can achieve highly repeatable, highly specific and highly sensitive detection of oropeceutical virus.

[0033] Although the embodiments of the present invention are described with reference to actual solutions, they do not constitute a limitation on the meaning of the present invention. Modifications to the embodiments and combinations with other solutions based on this specification will be obvious to those skilled in the art.

Claims

1. A primer and crRNA for detecting oropic virus nucleic acid, comprising a primer and crRNA, characterized in that, The primers include a forward primer and a reverse primer, and the base sequences of the forward primer, the reverse primer, and the crRNA are represented as follows: The forward primer: OROV-F:5'-TAATACGACTCACTATAGGGTGCTGAGATGTTTCTGCAGACATTCACTTTC-3', SEQ ID NO: 1, its position in GenBank reference sequence KP691605.1 is 479~509; The reverse primer: OROV-R: 5'-TGCTCTTACAGCAACTATTTCTTCACGCATCC-3', SEQ ID NO: 2, its position in GenBank reference sequence KP691605.1 is 616~647; The crRNA: 5-gauuuagacuaccccaaaaacgaaggggacuaaaacGCAGUCUUCUGGCCAUACCUCUGCCGAA-3', SEQ ID NO: 3, is located at position 580~607 in GenBank reference sequence KP691605.

1.

2. A kit comprising the primers and crRNA for oropeczema nucleic acid detection as described in claim 1, characterized in that: The kit includes RPA reaction reagents and CRISPR reaction reagents; The RPA reaction reagent includes buffer A, 20 μM primer OROV-F, 20 μM primer OROV-R, starter, and reaction tube, wherein primer OROV-F is the nucleotide sequence shown in SEQ ID NO: 1, and primer OROV-R is the nucleotide sequence shown in SEQ ID NO: 2; The CRISPR reaction reagents include Cleavage Buffer, Trans Mix, T7 RNA Polymerase, Cas13aProtein (2uM), crRNA (0.4uM), Reporter (4uM), and Nuclease-free Water, wherein the crRNA is the nucleotide sequence shown in SEQ ID NO:

3.

3. The reagent kit according to claim 2, characterized in that: The kit includes RPA reaction reagent and CRISPR reaction reagent, with a positive control being an RNA fragment transcribed from oropecie virus in vitro.

4. The reagent kit according to claim 2, characterized in that: The negative control was sterile physiological saline, which was extracted simultaneously with the sample during nucleic acid extraction to serve as a negative control; the blank control was ultrapure water without nuclease.

5. The reagent kit according to claim 3, characterized in that: The method for preparing the positive control includes the following steps: Step A1: Synthesize plasmid based on reference sequence KP691605.1 downloaded from GenBank; Step A2: Use T7 RNA polymerase to perform in vitro transcription to obtain single-stranded RNA, and then digest with DNase I to remove the DNA molecules. Step A3: Perform column purification using the QIAGEN RNeasy MiniElute Cleanup kit; Step A4: Determine the concentration of purified RNA using a micro-ultraviolet spectrophotometer, and calculate the copy number based on its molecular weight; Step A5: After aliquoting, store at -80℃ as a positive control for the kit.

6. A detection method comprising the kit of claim 3, characterized in that: The detection method includes the following steps: Step B1: Extract RNA from the sample, i.e. template RNA. The kit does not provide RNA sample extraction reagents. Users should select the appropriate commercial kit to extract viral nucleic acid according to the sample type. Step B2: Prepare the RPA reaction solution as follows: The reaction volume is 50 μL; add the reaction mixture to each reaction tube containing 29.4 μL of buffer A, 0.4 μM of each primer (OROV-F, OROV-R), 2.5 μL of starter, and 10 μL of RNA template; mix well, centrifuge for a few seconds, and place in a water bath, metal bath, or PCR instrument at 39℃ for 10 min; Step B3: Prepare CRISPR reaction solution for instrument detection: The reaction volume is 20 μL; the mixture contains 2 μL Cleavage Buffer, 4 μL Trans Mix, 0.5 μL T7 RNA Polymerase, 0.6 μL Cas13a Protein (2 μM), 1.5 μL crRNA (0.4 μM), 0.6 μL Reporter (4 μM), 5 μL of the product from the first reaction, and 5.8 μL Nuclease-free Water. After mixing, place the mixture on a fluorescence PCR instrument at 37℃ for 25 min, collecting the fluorescence of the FAM channel once per minute. Step B4, Result Judgment: First, the quality control system should be judged, that is, the amplification curve of the positive control in each fluorescence channel should show a standard S-shaped curve, while the negative control and blank control should not show a typical S-shaped curve. When the quality control system is effective, the results of the test samples are judged as follows: if the test sample has a clear S-shaped curve in the FAM channel, it is judged as positive for oropeceutical virus nucleic acid; if the test sample has no fluorescence amplification in the FAM channel, it is judged as negative for oropeceutical virus nucleic acid.

7. The detection method according to claim 6, characterized in that: The detection method includes sensitivity analysis. The method for sensitivity analysis is as follows: (1) Sample processing: Using the viral RNA transcribed in vitro as the template for detection, the concentration of purified RNA was determined using a micro-ultraviolet spectrophotometer. The copy number of the initial RNA template was calculated based on the molecular weight. Then, it was serially diluted 10-fold to single-digit copy numbers, for a total of 5 gradients. The copy number was 9 × 10⁻⁶. 2 copies / μL~10 -1 copies / μL; (2) Detection: The reaction solution is prepared and detected using the detection method of claim 6, and the minimum template copy number that the kit can detect is analyzed; (3) Results: The test results show that the detection limit of the kit is 9 copies / μL and the total detection time is 40 min. Therefore, the kit has a fast detection speed and high sensitivity, and can play an important role in rapid emergency detection.

8. The detection method according to claim 6, characterized in that: The detection method includes repeatability analysis. The method for repeatability analysis is as follows: (1) Sample processing: The viral RNA transcribed in vitro was used as the template for detection at a concentration of 9 copies / μL; (2) Detection: The reaction solution was prepared using the detection method described in claim 6, and the detection was repeated 10 times. Then, the reaction tube was placed on a fluorescence PCR instrument for detection. After the reaction was completed, the fluorescence curve was observed, and the repeatability of the detection by the kit was analyzed. (3) Results: The test results showed that the kit was positive in 10 replicates at a concentration of 9 copies / μL, which met the expected results. It can be seen that the test kit has good repeatability.

9. The detection method according to claim 7, characterized in that: The detection method includes specificity analysis. The method for the specificity analysis is as follows: Sample processing: A group of clinically positive samples of bloodborne infectious disease-associated pathogens were selected, including dengue virus type I, dengue virus type II, dengue virus type III, dengue virus type IV, chikungunya virus, Zika virus, yellow fever virus, monkeypox virus, Plasmodium falciparum, and Plasmodium vivax, plus in vitro transcribed oropecchiovirus RNA (10 4 (copies / μL) Testing: Using the test kit described above, the 11 samples were tested using the method described in claim 7, and it was observed whether the test kit would produce non-specific test results. Results: Analysis of the fluorescence spectrum amplified by the detection kit showed that the kit was positive only for samples containing oropic virus RNA, while the other 10 pathogens and the negative control were negative, demonstrating that the method has good specificity.

10. The detection method according to claim 8, characterized in that: Sensitivity and repeatability analysis of a kit for detecting oropic virus based on RPA-CRISPR / Cas13a technology using a visual results interpretation method: (1) Sample processing: Copy number: 9 × 10 2 copies / μL~10 -1 OROV RNA at a concentration of copies / μL was used as a template; (2) Detection: Using the kit, place the CRISPR reaction tube in a 37°C constant temperature water bath for 25 min. Finally, observe the color and interpret the result under 470nm blue light. Yellow-green fluorescent tubes are positive, and those without fluorescence are negative. Repeat the test 20 times with the lowest detection concentration of the template to determine its repeatability. (3) Results: According to the detection results, the lowest concentration of the sample emitting yellow-green fluorescence was 9 copies / μL, and all 20 repeated tests were positive; indicating that the detection limit of the kit under the visualization result interpretation method is 9 copies / μL. It can be seen that the detection kit is fast, highly sensitive, and does not depend on the fluorescence PCR instrument, and can play an important role in rapid emergency detection in areas with scarce resources.

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